AB0132 STIMULATION OF SOLUBLE GUANYLATE CYCLASE (sGC) FOSTERS ANGIOGENESIS AND BLUNTS ENDOTHELIAL-TO-MESENCHYMAL TRANSITION (EndoMT) OF SYSTEMIC SCLEROSIS (SSc) DERMAL MICROVASCULAR ENDOTHELIAL CELLS
Authors: E. Romano, I. Rosa, B. S. Fioretto, D. Giuggioli, M. Manetti, M. Matucci-Cerinic
Journal: Annals of the Rheumatic Diseases (2022)

Background In SSc, early abnormalities in microvessel morphology and angiogenic impairment in parallel advance with the development of tissue fibrosis orchestrated by myofibroblasts. Increasing evidence suggests that the EndoMT process, in which endothelial cells transdifferentiate into profibrotic myofibroblasts, may take centre stage in SSc pathogenesis [1,2]. sGC is an enzyme regulating cell growth/proliferation and vascular tone/remodelling by catalysing the production of cyclic guanosine monophosphate. Previous studies reported that sGC stimulation inhibits TGFβ-induced fibroblast-to-myofibroblast differentiation and collagen synthesis by blocking non-canonical ERK-dependent TGFβ signalling, and that sCG stimulators (sGCS) may exert antifibrotic effects in experimental models of fibrotic disorders. Objectives To investigate the possible modulatory effects of sGC stimulation on impaired angiogenesis and EndoMT of SSc dermal microvascular endothelial cells (SSc-MVECs). Methods To evaluate the effects of treatment with sGCS on endothelial cell viability/proliferation, 5 lines of SSc-MVECs and 5 lines of healthy dermal MVECs (H-MVECs) were challenged with sGCS (here MK-2947) and assayed with both annexin V/PI flow cytometry and WST-1. To analyse the modulation of angiogenesis by sGCS, SSc-MVECs were challenged with MK-2947 and subsequently tested for wound healing and capillary-like tube formation capabilities. To study the effects of MK-2947 on EndoMT, the same cells were assayed for the expression of endothelial and mesenchymal/myofibroblast markers by quantitative real-time PCR, western blotting and immunofluorescence, as well as for their contractile ability by collagen gel contraction assay. Phosphorylation of ERK1/2 was assessed by western blotting. Results Treatment with MK-2947 did not affect viability/proliferation of H-MVECs, while it significantly increased the proliferation of SSc-MVECs (p&lt;0.001 vs. basal). Compared to basal condition, the MK-2947 challenge ameliorated both wound healing capability (p&lt;0.001) and angiogenic performance (number of nodes: p&lt;0.01; segments: p&lt;0.001; meshes: p&lt;0.01; and junctions: p&lt;0.001) of SSc-MVECs. Upon stimulation of sGC, SSc-MVECs exhibited increased gene expression of proangiogenic matrix metalloproteinase (MMP)-9 (p&lt;0.05) and decreased expression of both antiangiogenic MMP-12 (p&lt;0.05) and pentraxin-3 (p&lt;0.001) respect to basal SSc-MVECs. A significant increase in both gene and protein expression of the endothelial markers CD31 and VE-cadherin, and a parallel decrease in the expression of the mesenchymal/myofibroblast markers α-SMA, S100A4, and type I collagen were found in MK-2947-treated SSc-MVECs. MK-2947 also downregulated the EndoMT-driving transcription factor SNAIL1 in SSc-MVECs. Stimulation with MK-2947 was able to significantly counteract the intrinsic ability of myofibroblast-like SSc-MVECs to contract collagen gels (p&lt;0.001) and effectively reduce phosphorylated-ERK1/2 protein levels (p&lt;0.01) respect to basal cells. Conclusion Stimulation of sGC effectively ameliorates the angiogenic performance and blunts the pathogenic myofibroblast-like profibrotic phenotype of SSc-MVECs. References [1]Manetti M, et al. Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis. 2017;76:924–34. [2]Romano E, et al. New insights into profibrotic myofibroblast formation in systemic sclerosis: when the vascular wall becomes the enemy. Life (Basel). 2021;11:610. Disclosure of Interests Eloisa Romano: None declared, Irene Rosa: None declared, Bianca Saveria Fioretto: None declared, Dilia Giuggioli: None declared, Mirko Manetti Speakers bureau: has received consulting fees or honorarium from MSD, Marco Matucci-Cerinic Speakers bureau: has received consulting fees or honorarium from Actelion, Janssen, Inventiva, Bayer, Biogen, Boehringer, CSL Behring, Corbus, Galapagos, Mitsubishi, Samsung, Regeneron, Acceleron, MSD, Chemomab, Lilly, Pfizer, Roche, Grant/research support from: has received consulting fees or honorarium from Actelion, Janssen, Inventiva, Bayer, Biogen, Boehringer, CSL Behring, Corbus, Galapagos, Mitsubishi, Samsung, Regeneron, Acceleron, MSD, Chemomab, Lilly, Pfizer, Roche